The present invention is directed to a method and a kit for amplifying nucleic acid segments and detecting nucleic acid analyte in a test sample. More specifically, the present invention relates to amplifying nucleic acid segments using the DNA-dependent RNA polymerase activity of RNA-dependent RNA replicases, such as Qxcex2 replicase, and detecting the products of such amplification.
The ability to detect specific target nucleic acid analytes using nucleic acid probe hybridization methods has many applications. Among these applications are diagnoses of infectious or genetic diseases or cancer in humans or other animals; identification of viral or microbial contamination of cosmetics, food or water; and identification or characterization of, or discrimination among, individuals at the genetic level, for forensic or paternity testing in humans and breeding analysis and stock improvement in plants and animals. The basis for applications of nucleic acid probe hybridization methods is the ability of an oligonucleotide or nucleic-acid-fragment probe to hybridize, i.e., form a stable, double-stranded hybrid through complementary base-pairing, specifically with nucleic acid segments which have a particular sequence and occur only in particular species, strains, individual organisms or cells taken from an organism.
One of the basic limitations in nucleic acid probe hybridization assays has been the sensitivity of the assays, which depends on the ability of a probe to bind to a target molecule and on the magnitude of signal that is generated from each probe that binds to a target molecule and that can be detected in a time period available for detection. Known detection methods in the assays include methods dependent on signal generated from a probe, as from fluorescent moieties or radioactive isotopes included in the probe, or an enzyme, such as an alkaline phosphatase or a peroxidase, linked to the probe and, after probe hybridization and separation of hybridized from unhybridized probe, incubated with a specific substrate to produce a characteristic colored product. However, the practical detection limit of these assays is about 200,000 target molecules (3 femtomolar concentration in 100 xcexcl), which is not sufficiently sensitive for many applications. Much effort is therefore being expended in increasing the sensitivity of detection systems for nucleic acid probe hybridization assays.
A second area of research which is receiving significant attention is enhancement of sensitivity by, in effect, increasing the number of target molecules to be detected, i.e., by the amplification of a segment of target nucleic acid to quantities sufficient to be readily detectable using currently available signal-producing and signal-detection methods. The traditional method of obtaining increased quantities of target molecules in a sample has been to grow an organism with the target molecule under conditions which enrich for the organism using various culturing methods. (Lennette, E. H., et al. (1985), Manual of Clinic Microbiology, editors, American Society for Microbiology, Washington, D.C.; Gerhardt, P., et al. (1981), Manual of Methods for General Bacteriology, Editors, American Society for Microbiology, Washington, D.C.). Recent advances in increasing the number of target molecules in a sample have focused on target-dependent increases in the number of reporter molecules which can be derived from individual target molecules. Such a xe2x80x9creporter moleculexe2x80x9d may or may not have the sequence of a segment of the corresponding target molecule. One example of these recent advances is amplification using the so-called xe2x80x9cpolymerase chain reactionxe2x80x9d (xe2x80x9cPCRxe2x80x9d). With respect to PCR amplification, reference is made to Current Protocols in Molecular Biology, Suppl. 4, Section 5, Unit 3.17, which is incorporated herein by reference, for a basic description of PCR. Other references which describe PCR include Erlich, H. A., (Ed.) 1989, PCR Technology, Stockton Press; Erlich, H. A., et al. (1988), Nature 331:461-462; Mullis, K. B. and Faloona, F. A. (1987), Methods in Enzymology, 155:335-350; Saiki, R. K., et al. (1986), Nature 324:163-166; Saiki, R. K., et al. (1988), Science 239:487-491; Saiki, R. K., et al. (1985), Science 230:1350-1354; U.S. Pat. No. 4,683,195 to Mullis, et al.; and U.S. Pat. No. 4,683,202 to Mullis.
In PCR, the double-stranded target nucleic acid is thermally denatured and hybridized with a pair of primers which flank the double-stranded segment of interest in the target (one primer hybridizing to the 3xe2x80x2-end of each strand of this double-stranded segment) and then the primers are extended in a DNA polymerase-catalyzed extension reaction. Numerous (e.g., typically twenty-five) cycles of the denaturation, hybridization and primer-extension process generate, for each target molecule in a sample of nucleic acids, many copies of reporter molecules, which are double-stranded DNAs with the same nucleic acid sequence as a segment (usually of about 100-2000 base pairs) of the target molecule. In a twenty-five cycle PCR amplification, more than about 106 reporter segments can be generated for each target molecule present initially in a sample. The PCR process is cumbersome because of the need to perform many cycles of the reaction, which usually require two or more hours for sufficient amplification. Additionally, the amplification process is more time-consuming if it is carried out manually. Further, it can be quite expensive if automated equipment is used.
Another recently disclosed amplification process, called the xe2x80x9ctranscription-based amplification systemxe2x80x9d (xe2x80x9cTASxe2x80x9d), uses primers which comprise segments for a promoter, which is recognized specifically by a DNA-dependent RNA polymerase which can produce quickly a large number of transcripts from segments operably linked for transcription to the promoter. Reference is made to Gingeras, T. R., et al., PCT Patent Publication No. WO 88/10315. Using suitable primers and primer-extension reactions with a single-stranded target molecule (e.g., an RNA or one strand of a double-stranded DNA) generates a double-stranded product which has a promoter operably linked for transcription to a pre-selected segment of the target molecule. Transcription of this product with a DNA-dependent RNA polymerase that recognizes the promoter produces, in a single step, 10 to 1,000 copies of an RNA comprising a sequence complementary to that of the target segment (i.e., the preselected segment of target molecule). Two additional rounds of primer extension using a reverse transcriptase enzyme and the RNA copies made in the initial transcription step produce CDNA copies which are ready for additional amplification by transcription using the DNA-dependent RNA polymerase to yield RNA with the same sequence as the target segment of target molecule. Additional cycles of CDNA synthesis and transcription can be performed. While TAS amplification, like PCR, makes a large number of reporter molecules (RNA in the case of TAS), which have the same sequence as a segment of the target molecule or the sequence complementary thereto, and uses fewer steps than PCR to achieve the same level of amplification, TAS requires two more enzymatic reactions, i.e., DNA-dependent RNA polymerase-catalyzed transcription and reverse transcription, and one or two more enzymes (DNA-dependent RNA polymerase and, if not used for primer-dependent DNA extension, reverse transcriptase) than PCR. Additionally, no time savings in comparison with PCR is claimed.
A third amplification procedure, which entails a form of amplification of label attached to a probe rather than amplification of a segment or segments of target nucleic acid analyte, is based on the use of the Qxcex2 replicase enzyme and its RNA-dependent RNA polymerase activity. Reference is made to Blumenthal, T. and G. G. Carmichael (1979), Ann. Rev. Biochem. 48:525-548; PCT Patent Publication No. WO 87/06270 and U.S. Pat. No. 4,957,858 to Chu, B., et al.; Feix, G. and H. Sano (1976), FEBS Letters 63:201-204; Kramer, F. R. and P. M. Lizardi (1989), Nature 339:401-402; U.S. Pat. No. 4,786,600 to Kramer; Lizardi, P. M., et al. (1988), Biotechnology 6:1197-1202; and Schaffner, W., et al. (1977), J. Mol. Biol. 117:877-907 for a further description of this procedure. In the procedure, a replicative (sometimes referred to as xe2x80x9creplicatablexe2x80x9d) RNA molecule is covalently joined to a specific hybridizing probe (i.e., a single-stranded nucleic acid with the sequence complementary of that of a segment (xe2x80x9ctarget segmentxe2x80x9d) of target nucleic acid analyte in a sample). The probe may be a segment embedded within a recombinant replicative RNA or attached to one of the ends of a replicative RNA. The probe-replicatable RNA complex hybridizes (by means of the probe segment) to target nucleic acid analyte in a sample, and the probe-RNA complexes that have hybridized are then separated from those that have not, and the replicatable RNAs of the complexes that did hybridize to target are then (typically after separation from probe segment if probe segment was not embedded in the replicatable RNA) amplified exponentially by incubation with Qxcex2 replicase, which catalyzes autocatalytic replication of the replicatable RNA to produce up to 109 reporter molecules (replicatable RNAS) for each hybridized target molecule. Such amplification can be completed in 30 minutes (Lizardi, et al., supra).
The extreme specificity of Qxcex2 replicase for RNAs with certain structural and sequence requirements for catalysis of autocatalytic replication assures that only the replicatable RNA associated with probes is amplified (Kramer and Lizardi, supra, 1989). Other advantages include the speed of the reaction and the simplicity of manipulations. However, a disadvantage includes the need to use RNA as a reporter molecule. An RNA of a given sequence is more expensive to manufacture and more sensitive to heat-stable nucleases than the DNA with the same sequence. In addition, except in cases where a probe segment can be embedded in a replicative RNA, the target segment is not amplified with the reporter molecules.
The present invention rests on the discovery of a DNA-dependent RNA polymerase (xe2x80x9cDDRPxe2x80x9d) activity of Qxcex2 replicase, the enzyme which catalyzes replication of the genome of the bacteriophage Qxcex2, and functional equivalents thereof (e.g., other RNA-dependent RNA replicases that have DDRP activity). The discovery of this DDRP activity allows the use of substrates which comprise 2xe2x80x2-deoxyribonucleotides or analogs thereof, including DNA substrates, for amplification by Qxcex2 replicase and the other replicases with DDRP activity.
The DDRP activity of an RNA replicase results in production of an RNA (or a polyribonucleotide in which, at some positions, ribonucleotide analogs are present), that is autocatalytically replicatable by the RNA replicase, from any substrate, which comprises a segment with the sequence of the autocatalytically replicatable RNA and which includes, within the segment with this autocatalytically replicatable sequence, a 2xe2x80x2-deoxyribonucleotide or an analog thereof, such as a 2xe2x80x2-deoxyriboalkylphosphonate, 2xe2x80x2-deoxyribophosphorothioate, 2xe2x80x2-deoxyribophosphotriester, or 2xe2x80x2-deoxyribophosphorami-date. In a substrate for the DDRP activity of an RNA replicase, the segment, which acts as the template for synthesis, catalyzed by the replicase, of the auto-catalytically replicatable RNA, can be a segment which encompasses the entire substrate (and, therefore, includes both the 3xe2x80x2-end and the 5xe2x80x2-end of the substrate), a segment which includes the 3xe2x80x2-end but not the 5xe2x80x2-end of the substrate, a segment which includes the 5xe2x80x2-end but not the 3xe2x80x2-end of the substrate, or a segment embedded within the substrate (and, therefore, including neither the 3xe2x80x2-end nor the 5xe2x80x2-end of the substrate). The substrate can be linear or closed circular and may be part of a double-stranded nucleic acid. The segment or the substrate may consist entirely of 2xe2x80x2-deoxyribonucleotides (i.e., a DNA segment or substrate, respectively). The substrates with which the DDRP activity is operative are not limited to homopoly-2xe2x80x2-deoxyribonucleotides, such as poly-das, with poly-dC segments at their 3xe2x80x2-ends, or RNAs with poly-dC segments at their 3xe2x80x2-ends. See Feix and Sano (1976), supra.
In the methods of the present invention, the substrates for the DDRP activity of RNA replicases are xe2x80x9ccomplexxe2x80x9d substrates. A xe2x80x9ccomplexxe2x80x9d substrate is one which is a closed circle, which does not have a free 3xe2x80x2-end; or one which has a free 3xe2x80x2-end but wherein the segment, which is the template for synthesis of an autocatalytically replicatable RNA catalyzed by the DDRP activity, does not include the 3xe2x80x2-end; or one which has a free 3xe2x80x2-end and wherein the segment, which is the template for synthesis of an autocatalytically replicatable RNA catalyzed by the DDRP activity, includes the 3xe2x80x2-end but has a segment other than a poly-dC at the 3xe2x80x2-end; or one which has a free 3xe2x80x2-end and wherein the segment, which is the template for synthesis of an autocatalytically replicatable RNA catalyzed by the DDRP activity, includes the 3xe2x80x2-end and has a poly-dC at its 3xe2x80x2-end but has, as the subsegment of said segment, other than the poly-dC at the 3xe2x80x2-end, a subsegment which comprises at least one 2xe2x80x2-deoxyribonucleotide or analog thereof but is not an homopoly-2xe2x80x2-deoxyribonucleotide. The segment, which is the template in a complex substrate for synthesis of an autocatalytically replicatable RNA catalyzed by the DDRP activity of an RNA replicase, is referred to as a xe2x80x9ccomplex segmentxe2x80x9d or xe2x80x9ccomplex template.xe2x80x9d In the methods of the invention, the xe2x80x9ccomplex segmentsxe2x80x9d comprise at least one 2xe2x80x2-deoxyribonucleotide or analog thereof.
Reference herein to a xe2x80x9cpoly dCxe2x80x9d means a segment of at least two dC""s.
Reference herein to a xe2x80x9c2xe2x80x2-deoxyribonucleotidexe2x80x9d means one of the four standard 2xe2x80x2-deoxyribonucleotides.
Reference herein to a xe2x80x9c2xe2x80x2-deoxyribonucleotide analogxe2x80x9d means an analog of a 2xe2x80x2-deoxyribonucleotide, which analog (i) has, as the base, the base of the 2xe2x80x2-deoxyribonucleotide or said base derivatized at a ring carbon or an amino nitrogen; and (ii) is other than the corresponding, standard ribonucleotide (rA for dA, rC for dC, rG for dG, U for T). A 2xe2x80x2-deoxyribonucleotide analog, that is part of a template for DDRP activity by an RNA replicase in accordance with the invention, will be recognized by the replicase in the template to place the ribonucleotide with the base, that is complementary to that of the 2xe2x80x2-deoxyribonucleotide, in the corresponding position of the autocatalytically replicatable RNA made from the template via the DDRP activity.
The RNA (or polyribonucleotide with one or more ribonucleotide analogs) made as a result of the DDRP activity of an RNA replicase is autocatalytically replicatable by the replicase (or another RNA-dependent RNA replicase which recognizes the RNA copies as templates for autocatalytic replication). Thus, a segment that is a template for the DDRP activity of an RNA replicase, is amplified, in the presence of the replicase, the ribonucleoside 5xe2x80x2-triphosphates, and, possibly, analogs of certain of the ribonucleoside 5xe2x80x2-triphosphates, because RNA (or polyribonucleotide with one or more ribonucleotide analogs) that is made in the synthesis catalyzed by the DDRP activity is autocatalytically replicated by the same replicase.
In its most general sense, then, the invention is a method for amplifying complex nucleic acid templates using the DNA-dependent RNA polymerase activity of RNA replicases, such as that of bacteriophage Qxcex2. The invention also entails numerous applications of this amplification method in making, amplifying, detecting, sequencing or otherwise treating a nucleic acid of interest. Thus, the amplification process can be used to make large amounts of RNA, which, for example, can be used as a nucleic acid probe, converted to cDNA for cloning, detected as part of a nucleic acid probe hybridization assay, or sequenced.
The amplification method of the invent-ion can be employed with a sample of nucleic acid in a target-dependent manner, such that an autocatalytically replicatable RNA which has, or comprises a segment with, a pre-determined sequence will be produced at a level detectable above background in the amplification carried out with the sample only if a xe2x80x9ctargetxe2x80x9d segment of nucleic acid (i.e., a segment with a pre-determined xe2x80x9ctargetxe2x80x9d sequence) is present in the sample. Thus, the invention entails a method for target nucleic acid segment-directed amplification of a reporter nucleic acid molecule which comprises using the DDRP activity of Qxcex2 replicase, or another replicase having DDRP activity. More specifically, to a sample of nucleic acid, one or more nucleic acid probes are added and the sample with the probes is processed such that a complex substrate for the DDRP activity of an RNA replicase, such as Qxcex2 replicase, occurs if and only if a target nucleic acid comprising one or more target segments occurs in the sample. This complex substrate is, or comprises, a complex segment which, in turn, comprises a pre-determined sequence (which is or comprises a reporter sequence) and which is the template for the DDRP activity. Each of the probes will hybridize to a target segment or the complement of a target segment and the probes will comprise segments such that, upon suitable processing, a nucleic acid that is the complex substrate for DDRP activity can be made using the probes if and only if the target segment(s) is (are) present in the sample. Once the sample has been treated so that substrate for DDRP activity occurs, if target nucleic acid is present, an RNA replicase, which has such activity with the substrate, is added, along with other reagents necessary for reactions catalyzed by the replicase, to an aliquot of the sample. If target nucleic acid was present, so that substrate for the DDRP activity was produced, autocatalytically replicatable RNA, which will have or comprise the reporter sequence or the sequence complementary thereto, will be amplified to detectable levels by the DDRP activity coupled with the autocatalytic replication of the RNA made with the DDRP activity. If target nucleic acid was not present, no substrate for the DDRP activity will be produced which comprises the reporter sequence and the replicase will not yield autocatalytically replicated RNA with such reporter sequence (or the sequence complementary thereto). RNA with such reporter sequence serves as a xe2x80x9creporterxe2x80x9d directly or, if further processed, indirectly. Thus, production of the RNA constitutes amplification of a reporter molecule and the process is target-directed (i.e., target-dependent).
The present invention provides methods for detecting the presence or absence of a target nucleic acid analyte in a sample containing nucleic acid. These methods of the invention comprise target-directed amplification in accordance with the invention, with the DDRP activity of Qxcex2 replicase, or other replicase with DDRP activity, of reporter nucleic acid, and assay for reporter nucleic acid.
Among advantages provided by certain of the methods according to the invention for detecting nucleic acid analyte is a reduction in the frequency of xe2x80x9cfalse positivesxe2x80x9d that occur in assays that employ such methods of the invention in comparison with assays that employ other methods. This advantage is associated with the fact that DNA is amplifiable (more precisely, capable of initiating amplification) using the DDRP activity of Qxcex2 and other replicases in connection with assay systems, and DNAs can be modified in specific ways using enzymes which do not modify RNAs in the same ways, if at all.
Several different embodiments of the target-dependent amplification methods of the invention are provided. These embodiments depend on different structures of, and methods of treating, the various nucleic acid probes employed to provide in a sample of nucleic acid, in a manner dependent on the presence of target nucleic acid in the sample, a complex nucleic acid segment which is amplifiable using the DDRP activity of an RNA replicase.
In one embodiment, to a sample of nucleic acid, which may include nucleic acid comprising a pre-selected target segment, a nucleic acid probe is added which comprises both a replicase-amplifiable, complex segment, which, as indicated above, comprises at least one 2xe2x80x2-deoxyribonucleotide, and an anti-target (xe2x80x9cprobingxe2x80x9d) segment, which has the sequence complementary to that of the target segment. The nucleic acid probe that hybridizes to target segment, if any, in the sample is separated from probe that did not hybridize and hybridized probe is treated under amplification conditions in the presence of Qxcex2 replicase, or another replicase exhibiting a DDRP activity with the replicase-amplifiable segment of the probe, resulting in the target-dependent production and amplification of reporter nucleic acid molecules. The process may also include the step of determining whether amplification has occurred.
In another embodiment, similar to that just described, the separation of probe hybridized to target from that not so hybridized is accomplished by subjecting the nucleic acid of the sample, after hybridization of probe to any target that may be present, to the action of a nuclease that will digest the replicase-amplifiable segment of any unhybridized probe. In this embodiment, in which probe, if hybridized, is protected from digestion, as in other embodiments of target-dependent amplification processes in accordance with the invention, if the amplification process is part of an assay for target analyte, amplified material will be tested for using any of the many methods known to the skilled.
In another embodiment, the target nucleic acid segment is hybridized with two probes in such a fashion that, after hybridization with the target nucleic acid, the 3xe2x80x2-end of the anti-target segment of one of the probes will be adjacent to the 5xe2x80x2-end of the anti-target segment of the other probe. Each probe comprises a portion of a nanovariant DNA, or other amplifiable DNA, covalently linked to anti-target segment. In one probe, the anti-target segment is at the 5xe2x80x2-end and, in the other, at the 3xe2x80x2-end. Once hybridized, the probes may be ligated via the anti-target segments. Preferably, T4 DNA ligase or another suitable ligase is used for the ligation. After the ligation, if it is carried out, or the hybridization, if ligation is not carried out, the adjacent probes are amplified via the DDRP activity of Qxcex2 replicase or a functional equivalent thereof. If the ligation/amplification process is, for example, part of a nucleic acid probe hybridization assay method, then, once amplification has been carried out, the amplified material is detected by a suitable means known to those skilled in the art. The amplified RNA, which comprises the sequence of the joined anti-target segments or the complement of that sequence, is a recombinant autocatalytically replicatable RNA wherein a segment, corresponding to the joined anti-target segments, is inserted into another RNA which is autocatalytically replicatable. Only if target segment was present in the sample will amplified RNA, which comprises the sequence of the joined anti-target segments or the complement of that sequence, be produced in the amplification process.
Two DNA probes are also employed in another embodiment of the invention. A first probe, for use in accordance with the embodiment, has a 3xe2x80x2-end which is an anti-target segment complementary (or nearly complementary) in sequence to a first target segment of target nucleic acid and is suitable for priming DNA synthesis using target nucleic acid as template. A second probe, for use in the embodiment, has a 3xe2x80x2-end which is a segment (termed a xe2x80x9ctarget-likexe2x80x9d segment) with the same (or nearly the same) sequence as a second target segment of target nucleic acid and also is suitable for priming DNA synthesis using the complement of target nucleic acid as template. The 3xe2x80x2-terminal nucleotide of said second target segment is located 5xe2x80x2 from the 5xe2x80x2-terminal nucleotide of said first target segment. Thus, the second probe can prime DNA synthesis on the primer extension product of the first probe annealed to target nucleic acid. The 5xe2x80x2-ends of both of the probes are replicase amplifiable or parts of nucleic acid that is replicase amplifiable, e.g., 5xe2x80x2-end of a nanovariant (+) DNA at the 5xe2x80x2-end of the first probe and 5xe2x80x2-end of a corresponding nanovariant (xe2x88x92) DNA at the 5xe2x80x2-end of the second probe. In the amplification process, the first probe is annealed to target and extended and the resulting extension products are preferably strand-separated by thermal denaturation, or if target is RNA, may be strand-separated by treatment with an enzyme providing RNase H activity. To the strand of the extension product which comprises the first probe at the 5xe2x80x2-end, the second probe is annealed and extended. Subsequent to, or simultaneously with, extension of second probe, amplification is catalyzed with the DDRP activity of Qxcex2 replicase or equivalent. If, but only if, target nucleic acid or its complement is present in a sample of nucleic acid with which this dual primer extension/amplification process is carried out, amplified product will include nucleic acid which comprises (i) the complement of the anti-target segment of the first probe, (ii) the target-like segment of the second probe, and (iii) the same segment, if any, between the segments of (i) and (ii) as occurs between the two target segments in target nucleic acid. Thus, a nucleic acid probe hybridization assay method of the invention is provided by following the dual primer extension/amplification process by any conventional assay for amplified product which comprises these two, or three, segments.
In another embodiment of the invention, a probe can be employed which is referred to for convenience as an xe2x80x9cRNA probexe2x80x9d but which either consists entirely of ribonucleotides (and is an RNA probe) or comprises in its sequence a sufficient number of ribonucleotides to permit degradation with a ribonuclease or chemical treatment that degrades RNA but DNA, if at all, at a much slower rate. The RNA probe comprises an anti-target segment, which is complementary or nearly complementary in sequence to a target segment, which is at the 3xe2x80x2-end of target nucleic acid or a segment thereof, so that target segment can prime DNA synthesis on the RNA probe as template. At its 5xe2x80x2-end the RNA probe comprises a replicase amplifiable segment. The target nucleic acid is treated so that the 3xe2x80x2-end of the target segment is xe2x80x9cfree,xe2x80x9d i.e., its 3xe2x80x2-terminal nucleotide has a 3xe2x80x2-hydroxyl and is at the end of a nucleic acid and not covalently joined, except through its 5xe2x80x2-carbon, to another nucleotide. The free 3xe2x80x2-end of target segment is preferably provided by any conventional technique by treating target nucleic acid prior to annealing RNA probe to target (or part thereof). The probe, and any target in the system, are combined under hybridizing conditions, the target segment is extended in a primer-extension reaction catalyzed by the reverse transcriptase activity of an enzyme which has such activity, and the RNA in the system is then degraded chemically or using enzymes with RNase activities, as understood in the art. This degradation of RNA is sufficiently extensive, when coupled with dilution that might also be carried out, to diminish the concentration of RNA probe that retains a replicase-amplifiable segment and that thereby is operative, as a template for amplification by the replicase to be employed subsequently in the process, to a sufficiently low level that amplification of any such probe that might remain in an aliquot of sample on which amplification is carried out will not be observable. Typically, after the reverse transcription of the RNA probe, the solution (or an aliquot thereof) will be treated so that the concentration of amplifiable-segment-retaining RNA in the aliquot of carried out will be less than 1/1000, and preferably less than 1/10,000, the concentration of complex template for DDRP activity that will be present if target segment was present in the sample of nucleic acid to which the amplification process is applied. More preferably, degradation of RNA will be coupled with dilution so that, statistically, less than one molecule of RNA with an amplifiable segment remains in the aliquot on which amplification is carried out. Any DNA-extension product remaining after target segment extension and RNA degradation comprises a replicase-amplifiable, complex DNA segment. After degradation of RNA in the system, and substantial elimination of RNA-degrading conditions or activities, the DDRP activity of Qxcex2 replicase or a functional equivalent is employed to amplify the replicase amplifiable segment added to any target DNA. Amplification will occur only if target segment, capable of priming DNA extension reaction on RNA probe as template, was present in a sample being tested. Thus, by applying after the amplification reaction any conventional method to test for the presence of amplified product, a method of assaying for target nucleic acid is also provided.
An RNA probe, which may consist entirely of ribonucleotides or comprises in its sequence a sufficient number of ribonucleotides to permit degradation with a ribonuclease or chemical treatment that degrades RNA but not DNA, can be employed in another embodiment of the invention, wherein three probes are employed. The first probe, which can be DNA or RNA or chimeric (i.e., any combination of ribonucleotides and 2xe2x80x2-deoxyribonucleotides or analogs of either), comprises at its 5xe2x80x2-end a first anti-target segment with the sequence complementary to or nearly complementary to that of a first target segment of target nucleic acid. The first probe must hybridize to its corresponding target segment with sufficient stability to block chain-extension of a second probe, as presently described. The second probe, which also can be DNA or RNA or chimeric, compromises a second anti-target segment at its 3xe2x80x2-end with the sequence complementary to, or nearly complementary to, that of a second target segment of target nucleic acid and, when annealed to target nucleic acid, is capable of priming DNA synthesis, using target nucleic acid as template. The 3xe2x80x2-end of the first target segment is located 5xe2x80x2 from the 5xe2x80x2-end of the second target segment and is separated from the 5xe2x80x2-end of the second target segment by a gap of at least several, and up to about 2000, bases. The third probe is referred to for convenience as an RNA probe but, like the RNA probe described above, which comprises a replicase-amplifiable segment, must only comprise a sufficient number of ribonucleotides to be susceptible, through processes which degrade RNA, to having eliminated the replicase-amplifiability of its replicase-amplifiable segment. The third probe comprises a target-like segment at its 3xe2x80x2-end and a replicase amplifiable segment. The target-like segment has the same or nearly the same sequence as a third segment of target nucleic acid, which comprises at its 5xe2x80x2-end at least several nucleotides of the gap between the first and second target segments (and may overlap the second target segment) and which has as its 5xe2x80x2-terminal base the base that is adjacent to the 3xe2x80x2-terminal base of the first target segment. The third probe, through the target-like segment, must be capable of priming DNA synthesis using as template the chain-extension product of the second probe, made using target nucleic acid as template. To amplify a reporter segment in accordance with the invention, in a target nucleic acid-dependent manner, the nucleic acid of a sample is rendered single-stranded and first and second probes are added to the sample, which is subjected to conditions whereby the probes will anneal to target if present and second probe, once annealed, will be extended in a primer-directed, template-dependent DNA extension reaction catalyzed by an enzyme such as Klenow Fragment of E. coli DNA polymerase I. The extension added to second probe in this extension reaction will have the sequence complementary to that of the gap between the first and second target segments in target nucleic acid. After the extension reaction, the sample is treated to strand-separate (e.g., thermally denature) the extension product, and then subjected to conditions whereby the third probe anneals to its target segment, which will comprise at least part of the 3xe2x80x2-end of the segment added to the 3xe2x80x2-end of second probe in the extension reaction and may overlap at least a part of the segment of the extension product which was second probe, and at least the extended second probe is further extended, employing reverse transcriptase activity and the third probe, including its replicase amplifiable segment, as template. Subsequent to the second extension of second probe, the sample is treated as described above, for the embodiment of the invention which utilizes an RNA probe, to substantially eliminate replicase-amplifiable segment of third probe by diminishing the concentration of such segment to an insignificant level before replicase is added to effect amplification. Thus, the solution is subjected to conditions to degrade RNA chemically or enzymatically, as understood in the art, and might be treated further to dilute remaining replicase-amplifiable segment of third probe. After degradation of the RNA and substantial elimination of RNA-degrading conditions, Qxcex2 replicase or another RNA replicase, which recognizes the replicase-amplifiable segment of the RNA probe as a template for autocatalytic replication, is added to the sample and the sample is subjected to conditions whereby the DDRP activity of the replicase catalyzes amplification from the complex, replicase-amplifiable segment of doubly extended second probe. As in other embodiments of the invention, once the DDRP activity-catalyzed amplification has occurred, the amplified material may be detected by suitable means known to those skilled in the art.
As the skilled will understand, target segment(s) is (are) selected so that, in a sample of nucleic acid to which a method of the invention is applied, target segment, or the combination of target segments, required for amplification in accordance with the method of the invention to occur is present in an amount distinguishable from background only if target nucleic acid is present in the sample. Preferably target segment(s) is (are) selected so that the required target segment or combination of target segments is absent from a sample unless target nucleic acid is present.
The present invention is also directed to quantification of the amount of target nucleic acid analyte in a sample. Quantification is accomplished by comparing the amount detected of a first amplified nucleic acid, the amplification of which occurs only if target nucleic acid analyte is present in a sample, with the amount detected of a second amplified nucleic acid, the amplification of which is carried out in parallel with that of the first amplified nucleic acid and occurs on account of the presence in the sample of a preselected nucleic acid which serves as an internal standard and is present in the sample in a known amount.
The present invention also encompasses a test kit for detection of a specific target nucleic acid analyte in a sample of nucleic acid. The kit comprises one or more nucleic acid probes required for amplification, in accordance with the invention, of a reporter molecule, Qxcex2 replicase or an equivalent enzyme to provide DDRP activity, and other enzymes (if any) required for processing of analyte or probe(s) prior to or simultaneously with amplification catalyzed by the DDRP activity. The kit may also comprise means for detecting reporter nucleic acid produced in the amplification according to the invention, and various components, such as buffers and nucleoside triphosphates, to facilitate carrying out the required hybridizations and enzymatically catalyzed reactions, including autocatalytic replication. A kit may also comprise components required for amplification associated with a pre-selected nucleic acid as an internal standard and detection of product from such amplification, in order to provide for quantification in accordance with the invention of target nucleic acid analyte to be assayed for with the kit. The various components of kits according to the invention may be packaged in a kit in any of a variety of ways, among usually a plurality of vials or other containers, as dictated by factors understood in the art, such as the need to preserve the stability and purity of the components over the shelf-life of the kit, the order in which various components are used in accordance with the invention, convenience in using the kits, convenience and cost in manufacturing the kits, and the like.
Various methods known in the art can be employed to detect reporter molecules provided by an amplification process in accordance with the invention. Thus, the reporter nucleic acid can be reacted with various dyes and the dye detected visually or spectrophotometrically. Alternatively, a ribonucleoside 5xe2x80x2-triphosphate, that is labelled for detection and remains active as a substrate for the Qxcex2 replicase or other replicase catalyzing the amplification, can be employed in the amplification reaction and then signal from the label incorporated into the amplified reporter can be detected directly or, after association of the label with a signal-generating molecule, indirectly. In still another alternative, reporter nucleic acid resulting from amplification can be hybridized with nucleic acid probe that is labelled for detection and signal associated with such probe hybridized to reporter can be detected directly or indirectly.
Among the advantages of the methods of the invention, and kits of the invention for carrying out the methods, is speed. The amplification process of the invention is typically able to produce more than 109 reporter molecules for each target molecule in a sample in about 60 minutes. Further, systems for carrying out the present invention are relatively simple in design and superior to systems which require use of RNA to initiate amplification. The DNA used for this purpose in methods of the present invention is resistant to degradation catalyzed by RNases and provides more synthetic options than their RNA counterparts. Chemically synthesized DNA also provides a cost advantage over RNA. The present invention is especially useful for amplification based on a rare species of nucleic acid present in a mixture of nucleic acids to provide effective detection of the presence, and quantity, of the species.
Target nucleic acid analytes for amplification or detection by the methods of the present invention include, inter alia, nucleic acids characteristic of bacteria, viruses and other vectors of human infectious diseases; genomic nucleic acids comprising abnormalities which underlie human genetic diseases; genomic nucleic acids comprising human cancer genes; nucleic acids used in forensic analyses, paternity testing, compatibility testing for bone marrow transplantations, characterization of plants and animals using restriction fragment length polymorphism, and correlations of improvements through animal-or plant-breeding with genetic changes; and nucleic acids characteristic of organisms which contaminate foods, cosmetics or water or whose presence is diagnostic of environmental conditions in the environment in which the organisms occur.